Luciferase assay, compositions and kits for use in connection therewith

Abstract

An assay for the presence of luciferase in a biological sample offers heightened sensitivity, signal intensity and persistence. The assay is sensitive down to 50 fg luciferase. The biological sample is combined with essential ingredients luciferin, ADP, myokinase and Mg++. The myokinase converts ADP to ATP, necessary for the luciferase reaction, and AMP, which retards the reaction kinetics. The resulting assay exhibits a persistent glow emission which makes it adaptable to automation.

What is claimed is:  
     
       1. A method of assaying for the presence of luciferase in a biological sample, comprising: 
       combining said sample with a reaction composition which comprises luciferin, adenosine diphosphate (ADP) and myokinase;  
       allowing said reaction composition to incubate with said biological sample; and  
       inspecting said incubated biological sample and reaction composition to determine if light is emitted, wherein said light emission is indicative of the presence of luciferase in said biological sample and wherein the intensity of said light emission is a sustained glow that persists over an prolonged period of time to permit automated detection of the presence of luciferase in said sample.  
     
     
       2. The method of claim  1 , wherein said reaction composition further comprises a sulfhydryl luciferase stabilizing agent. 
     
     
       3. The method of claim  1 , wherein said reaction composition further comprises a source of Mg ++  ions. 
     
     
       4. The method of claim  2 , wherein said reaction composition further comprises a source of Mg ++  ions. 
     
     
       5. The method of claim  1 , wherein said reaction composition further comprises at least one buffering agent. 
     
     
       6. The method of claim  1 , wherein said reaction composition further comprises at least one of dextran and glycerol. 
     
     
       7. The method of claim  1 , wherein said reaction composition is formed by combining two reaction buffers, wherein said first buffer comprises myokinase, and said second buffer comprises ADP and luciferin and said first and second reaction buffer are combined to form said reaction composition. 
     
     
       8. The method of claim  7 , wherein said first reaction buffer further comprises a source of Mg ++  ions, and a sulfhydryl (SH) luciferase stabilizing reducing compound. 
     
     
       9. The method of claim  8 , wherein said SH compound is dithiothreitol, β-mercaptoethanol or mixtures thereof. 
     
     
       10. The method of claim  7 , wherein said first reaction buffer further comprises a zwitterionic buffering agent and glycerol, and said second reaction buffer comprises dextran.